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Parkinson disease susceptibility gene mutations in Drosophila melanogaster and the therapeutic potential of transgenes alternative oxidase and alternative NADH dehydrogenase

Final Report Summary - DROMIT (Parkinson disease susceptibility gene mutations in Drosophila melanogaster and the therapeutic potential of transgenes alternative oxidase and alternative NADH dehydrogenase)

Mitochondria are the powerhouses of the cell. The cell uses a universal energy unit, adenosine triphosphate (ATP), to carry out its functions, such as maintenance of ion homeostasis, muscle contraction, and neuronal signal transduction. In the mitochondria the energy contained in foods, such as carbohydrates and lipids, undergo the last steps of energy conversion into ATP. ATP is then transported out of the mitochondria via specific transporters and released into the cytosol to be consumed by the cell.
Equal distribution of the mitochondrial network throughout the cell is essential for cellular functions. This is most apparent in the neurons where the energy requirements are greater than in most other types of cells and the energy requirement peaks at sites of signal transduction. The mitochondria are distributed within the cell with an active energy-requiring process. This process responds to cellular cues, which are currently mostly unknown, assigning the mitochondria to areas in need of energy. The transport of mitochondria is carried out by the two cellular motor proteins, dynein and kinesin. These proteins attach themselves to the mitochondrial surface proteins via adapter proteins and then “walk” along the microtubules that run from the centriole of the cell to the cellular periphery.
In the current study we wanted to find factors that affect the cellular transport of mitochondria in the neurons. In our analysis, we used primary neurons derived from pupal stage Drosophila melanogaster. The Drosophila neurons were selected for this study in order to create a starting point for future studies in mitochondrial transport which could utilise the transgene manipulations available in the Drosophila. The mitochondria were visualised with green fluorescent protein (GFP) localised to the mitochondrial matrix. The parameters of mitochondrial movement were analysed separately for neuronal cell bodies (the soma) and neuronal processes (neurites) that were either proximal to the soma or distal to the soma.
We report that mitochondria move faster and have longer run lengths in the soma than in the aforementioned, distal and proximal, processes. As a possible explanation for this, we propose that the confinement in the process is causing resistance to the mitochondrial transport which results in a decrease in both run length and speed of transportation. In the neuronal processes the run length and velocity are not affected by the length of the process or the localization of the mitochondria in it, thus the resistance stays equal throughout the process.
Our hypothesis of resistance of transport induced by confinement was tested by treating the neuron cultures with a mildly hypotonic media. Analysis of mitochondrial transport under hypotonic conditions revealed increased velocity and run lengths of mitochondria in the processes. The confinement hypothesis was further tested by treating the neuron cultures with Latrunculin A, which depolymerizes actin, a cytoskeletal protein that is used to anchor the mitochondria in place. Again, the parameters of mitochondrial transport in Latrunculin A treated neurons were increased in the neuronal processes. Both treatments had minor effects on mitochondrial transport in the soma.
This study provides us with information on the biophysical properties affecting the mitochondrial transport in neurons. It will be used in forthcoming studies with genetic manipulations linked to human neurodegenerative diseases. Data gathered from this study may be used as a reference when researching mitochondrial transport in other types of neuronal cultures. Our results should also be taken into consideration in diseases where the disease is manifested with impaired mitochondrial transport such as Charcot-Marie-Tooth Disease.
In second project we were studying the genome wide gene-expression changes induced by mutation in the Drosophila mitochondrial ATP-adenosinediphosphate (ATP-ADP) transporter stress sensitive b (Sesb). The mutation in the ATP-ADP transporter, ANT1, in humans causes autosomal dominant progressive external ophthalmoplegia (adPEO), which causes myopathy, cardiomyopathy and exercise intolerance. The Drosophila Sesb1 mutation in the Sesb gene is homologous to disease causing mutation in the human ANT1 gene. When analysing genome wide gene expression in the Sesb1 mutant Drosophila, we found gene expression changes that were consistent with a metabolic shift towards glycolysis.
The Sesb1 mutant Drosophila females are infertile and in the gene-expression analysis a clear downregulation of genes required for oogenesis was observed. Interestingly, the eggs of the Sesb1 mutant Drosophila are fertilised and undergo embryo development, but fail to produce living larvae. In this study we were able to partially rescue the infertility phenotype by transgenic expression of alternative oxidase (AOX) from Ciona intestinalis. The rescue is considerable since normally the Sesb1 mutant females have zero adult progeny but with autosomal expression of AOX the viable adult progeny from Sesb1 mutant females was up to one fifth of that of the wild type females.
Other phenotypes related to Sesb1 mutation are characterized by delayed development and stress sensitivity. Both of these phenotypes were ameliorated by changing the mitochondrial DNA (mtDNA) to that which originated from flies that had been previously infected with endosymbiont bacteria and is thus considered to harbor beneficial mtDNA mutations. The stress sensitivity was also reduced when the expression of the key regulator of mitochondrial biogenesis PGC-1alpha was increased. This data provides evidence that pharmacological strategies aimed at boosting mitochondrial biogenesis, as well as genetic therapies based on AOX, could be effective treatments for mitochondrial myopathies and other human diseases associated with ANT1 deficiency.